Methods for the determination of alflatoxin B1 in feedingstuffs intended for dairy cattle

Afdeling Organische Contaminenten/ Bestrijdingsmiddelen 1984-10-10 RAPPORT 84.92 Pr.nr. 505.0421

Methods for the determination of

aflatoxine n1 in feedingstuffs intended for dairy cattel.

Verzendlijst: direkteur, sektorhoofden, direktie VKA, afd. OCON (4x), afd. Normalisatie/Harmonistie (Humme), Projektbeheer,

Projektleider.

7912003

RIJKS-KWALITEITSINSTITUUT VOOR LAND-EN TUINBOUWPRODUKTEN

WAGENINGEN

DRAFT 1 dd. 1984-09-01

METHODS FOR THE DETERMINATION OF AFLATOXIN B1 IN FEEDINGSTUFFS INTENDED FOR DAIRY CATTLE.

L.G.M.Th. Tuinstra and W.A. Traag

State Institute for Quality Control of Agricultural Products, Bornsesteeg 45, 6708 PD Wageningen, The Netherlands.

This report is written on request of the "Committee of Experts on Methods of Analysis of Feedingstuffs" (Commission of the European Com-munities, Directorate-General for Agricultural VI B 3).

Introduetion

Until the first of January 1984 within EC an aflatoxin _{B1 toleranee} of 20 ~g/kg was in force for feedingstuffs intended for dairy cattle (Directory 74/73/EEG dd. 17-12-1973) including a methad of analysis 76/372/EC dd. 1-3--1976).

This official methad has some disadvantages.

a) The limit of detection, using two dimensional thin layer chromato-graphy (TLC) is about 10 ~g/kg.

b) When citrus pulp is used as ingredi~nt the methad can nat be used. (Already in 1975 it was indicated in literature (1) that by

changing the composition of the TLC eluents separation between aflatoxin B1 and interfering compounds from citrus pulp could be improved and solve the problem.)

c) The methad is time consuming.

From the first of January 1984 the aflatoxin _{B1 toleranee beeame} 10 ~g/kg (74/63/EEG dd. 28-7-1983). In general it is desirabie that a methad of analysis has a limit of determination being 10-20% of the

toleranee. In other words with a toleranee of 10 )Jg/kg a determination limit of 1-2 ~g/kg should be realized meaning a detection limit of

< 1 ~g/kg.

As the methad used up till now has a detection limit in the order of the new toleranee another methad should be used.

During the 56th meeting of "the committee of experts on methods of analysis of feedingstuffs" (dd. 16-17 april 1984) it was deeided that

the several delegations should send methods of analysis to the RIKILT and '"hen possible aecompanied \<lith "data". As no other methods \o~ere received the list of methods shows only the methods made available during the above mentioned meeting.

From the German delegation a more detailed methad was received and some data (without relevant text) concerning ringtests.

In Annex 1-4 the methods are given; in Annex 5 the Dutch data and in Annex 6 the German data.

Below the principles of the four methods will be described.

-Principals of proposed methods

a. Proposal of the Netherlands (2,3,4) (Annex 1 and 5).

- Aflatoxin B1 is extracted with chloroform. For concentrated cattle feed an aliquot of 40 mg is cleaned \~ith thin layer chromatography on silica coated alumina plates.

The aflatoxin _D1 area is cut out of the plate and extracted deter-mination in the extract of 16 mg is carried out on a reversed phase HPLC column \üth a fluorescence detector after post column derivati

-zation at 60°C with an iodine water solution. Absolute limit of detection is about 0,02 ng.

- The methad is applicable to the determination of aflatoxin _D1 in concentrated cattle feed.

The limit of determination is 1 ~g/kg. Recovery at 5 ~g/kg is be-U-leen 80-100%.

For raw materials, without clean-up, the limit of determination is about 25 ~g/kg. Recovery is better than 80%.

b. Proposal of the United Kingdom (5) (Annex 2).

- Aflatoxin B1 is extracted with chloroform. An aliquot of 24 gram is cleaned by a disposable sep-pak silica cartridge.

Determination in the extract of 1 gram is carried out on a silica HPLC column '"i th a fluorescence detector. Absolute limit of

detec-tion is about 0,4 ng.

- The methad is applicable to the determination of aflatoxin _{B1 in} concentrated cattle feed. The limit of determination is about 0,4 ~g/kg.

c. Proposal of Germany (6) (Annex 3 and 6).

- _{Aflatoxin B1 is} extracted with chloroform. An aliquot of 10 gram is concentrated to dryness and solved in cyclohexane-ethylacetaat (1/1 v/v) and cleaned with gelpermeation chromatography on bio beads SX3. Determination in the extract of 2.4 gram is carried out with high per-formance thin layer chromatography (HPTLC) and densitometric quan-tification.

Absolute limit of detection is about 0,5 ng.

- The methad is applicable to the determination of aflatoxin Dl in concentrated cattle feed. The limit of determination is about 0.3 ~g/kg.

-- 3

-d) Proposal of Danmark (Annex 4).

- Aflatoxin _B1 is extracted with chloroform. An aliquot of 10 gram is cleaned on a silica column. Determination in the extract of 100 mg is carried out with a HPLC silica column \-lith a fluorescence detec-tor equiped with a packed flow-cell. Absolute limit of detection is about 0,05 ng.

- The method is applicable to the determination of aflatoxin B₁ in concentrated cattie feed. The limit of determination is 0.5 ~g/kg.

In table I a few important parameters are summarized. Table 1

Proposal of Amount of material Absolute Limit of

extracted cleaned applied detection ~g/kg

(g) ( g) (g) (ng) determination

The Netherlands 25 0,040 0,016 0,02 1

U.K. 50 10 1 0,4 0,4

Germany 50 24 2,4 0,5 0,3

Danmark 50 10 0,100 0,05 0,5

Discussion: Advantages and drawbacks

Ad A Dutch proposal

Experience shows that for a good performance of the metbod

con-siderable experience and skill is needed. The HPLC in combination \-lith the post column derivatization requires carefull adjustment of the different parameters. To achieve good repeatability the HPLC eluent and the derivatization reagent should be freshly made, flows should be checked regulary as well as the temperature of the water bath. When carrying out the clean-up on TLC plates dry ethylether must be used to avoid elution of af_{latoxin B1}• It is advised to dry the ether \-lith metallic sodium and to check this part of the clean-up by

cantrolling under UV light if aflatoxin _{B1 stayed at} the spot where it should be. In Annex 5 some data concerning reproducibility with in one lab is reported.

~ U.K. proposal

Experiments have shown that the capacity of sep-pak used for clean-up is rather limited and that separation between aflatoxin _{B1 and com} -pounds intertering with the HPLC method is rather poor.

-The combination of a silica sep pak and a silica HPLC column seems

questionable from a point of view of getting rid of intertering

com-pounds.

Further experience with a great scale of feedingstuffs of different composition seems desirable, with special attention to citrus pulp.

The use of silica HPLC columns has some drawbacks. The composition of the eluent is critica!; small changes of the water content influences

the activity of the column thus the separation and the sensitivity.

The reproducibility from day to day with respect of retention time and sensitivity 'olill be more difficult than ,.,ith reverse phase columns. Striking is the high flow (2,5 ml/min) used for the HPLC separation as described in the proposed method. (See also further remarks.)

Ad C German proposal

Experiments at the RIKILT with bio-beads SX3 GPC columns in the system

toluene-ethylacetaat (1:1) showed that when analysing citrus pulp con -taining feedingstuffs the fraction containing aflatoxin

n

_{1 also} con-tained strong fluorescing compounds (from the citrus pulp). On the other hand prof. Ranfft informed us that with his system (cyclohexan e-ethylacetaat 1:1) no compounds from citrus pulp were detectable on the TLC.

Into our apinion in the German proposal it should therefore be

necessary to describe how to optimize collecting the correct fraction.

Also should be indicated how much of the chloroform extract is used

for the GPC clean-up (and not as much as possible").

For the TLC determination the extract is applied in 200 ~1 ( 2,4 gram). It is believed that this procedure brings problems e.g. flow out of

the spot on the plate.

An improved description of the spatting technique is desirable. Though

the GPC technique can be automated and a high throughput of samples can be anticipated the literature (6) indicates however that one

tech-nician can handle only 5 samples a day. Annex 6 indicates good repro-ducibility.

-- 5

-Ad D Danish proposal

As this proposal is derived from the original EC method (76/372/EC dd. 1-3-1978) which excludes the method as suitable for citrus pulp

con-taining feedingstuffs and keeping in mind that HPLC separation on a silica column is not as good as a t~o10 dimensional TLC separation this proposal seems less suitable for routine analysis in a great variety of samples.

The use of a silica packed flow cell enhances the fluorescence of aflatoxin _B1 strongly. Experience however learned that in the time due

to adsorbing materials, the cell shows an increased background signal

influencing the gain in sensitivity in a negative way.

Problems mentioned with the reproducibility as indicated above for the

UK method are also here appropriate.

Further remarks

From the UK delegation information was received that the proposed UK method should be ringtested in the coming year. They confirmed their

experience with the Dutch method and were of the opinion that the

Dutch method was more appreciated than the own method.

For the time being and in expectation of the results of the UK ring-test with the UK method they had no objections to exclude the UK method from further discussion.

Recommendations

It seems advisable to focus attention to only two methods e.g. the Dutch and German proposal.

To gather information on repeatability and reproducibility a pre limi-nary interlaboratory study should be carried out.

May be it is advisable to look out for collaborating with the myco-toxin group of the BCR.

-Literature

1. L.G.M.Th. Tuinstra, C.A.H. Verhulsdonck, J.M. Bronsgeest, W.E.

Paulsch: Neth. J. Agric. Sci. 23 (1975) 10-17.

2. N.O. Davis, U.L. Diener: J.AOAC

&l

(1980) 107-109.

3. G.W. Thorpe, G.H. Hare, A.E. Pohland: Vth international IUPAC sym

-posium on mycotoxins and phytotoxins, Vienna, september 1-3, 1982, 52-55.

4. L.G.M.Th. Tuinstra, H. Haasnoot: Journal of Chromatography, 282

(1983) 457-462.

5. M.V. Howell, P.H. Taylor in J.AOAC Off. Anal. Chem., 1981, 64, no. 6 1356.

6. H. Nijhuis, H. Heeschen, C. MUhlhoff, Milchwissenschaft 38 (3) 1983.

Annex 1

Version d.d. 1984-09-01

The determination of aflatoxine B1 in feedingstuffs and raw materfals "'rlth HPLC and post column derivatization.

L.G.M.Th. Tuinstra,

w.

Haasnoot.

State Institute for Quality Control of Agricultural Products, Borns

e-steee 45, 6708 PD Wageningen, The Netherlands.

1. Purpose and scope

The methad is applicable to the determination of aflatoxin B1 in con

-centrateel cattle feed up to 1 ~g/kg. Recovery at 5 ~g/kg is between 80-100%. For ra"' materfals the methad is applicable, without clean-up up to 25 ~g/kg. Recovery is better than 80%.

2. Principle

Aflatoxin B1 is extracted witl1 chloroform. For concentrated cattle feed an aliquot is cleaned ~-1ith thin layer chromatography on silicagel coated alumina plates. The aflatoxin B1 area is cut out of the plate

and extracted. For raw materials an aliquot is just evaporated to dry

-ness and solveel in methanol.

Final separation and determination is carried out on a reverse phase

column. Post column derivatization at 60°C is carried out with a iodine ,.,a ter salution (1, 2).

3. Reagents

The reagents should be of such a quaU.ty that under the given analy-tica! circumstances no interference occurs. This should be tested.

3.1 Celite 545. (Johns Mansville) cat.no. 53999.

3.2 Chloroform, stahilizeel \dth 0,5-1% ethanol.

3.3 Aceton.

-5.2 Extraction

Take a 25 g test sample and introduce it in the 250 ml glass flask. Add 12.5 g celite, 12.5 ml water and 125 ml chloroform. Close the flask with the teflon lined rubber and screwcap and shake 30 min. Filter over fold filter and collect for cattle feed 5.0 ml in a cali-brated vial. Evaparate at 50°C under _{N2 stream to dryness. Dissolve}

the extract in 500 ~1 chloroform (using a vortex mixer) and use ex -tract for clean-up with thin layer chromatography.

(For raw materials after filtering collect 0.5 ml in a calibrated vial and continue as described under 5.4.)

Evaparate at 50°C under N2 stream to dryness. Dissolve the extract in 1 ml methanol (using a vortex mixer) and proceed as described under 5.3).

5.3 Clean up with thin layer chromatography.

5.3.1 Spatting

Draw with a pencil, according to fig. 2, lines on the plate. Spot at A, Band C _{1 ng aflatoxin B1} (= 10 ~1 of 0.1 ~g/ml).

Spot at S 20 ~1 of six different sample extracts (5.1).

5.3.2 Development of the plate

Develop with diethylether (remark 1) in a saturated tank in the first direction until the solvent front reaches the top of the plate (~ 45 min). Remave the plate from the tank, evaparate theether in the dark at room-temperature during 15 min. and cut the plate as indicated in fig. 2 (remark 2).

Develop the plate in an unsaturated tank with a mixture of chloroform: aceton:water

=

88:12:0.2 (v/v/v) in the opposite direction of the first development until the front reaches the top of the plate (~ 30 min.). Remave the plate from the tank and evaparate the solvents at roomtemperature in the dark.

Examine the plate under UV-light to localize the blue spots of the af-latoxin B1 standards. Mark these spots by drawing .5 cm above and be-low the aflatoxin _B1 standard a small line (see fig. 3).

-- 5

-Draw a line with a pencil connecting the three marks above and below the aflatoxin n₁ standard. Cut for every one of the six samples, the

parts from the plate. Each part is put into a separate test tube. Add 4 ml of dich1oromethane/aceton (6/4 v/v), mix with vortex mixer. Filter the dich1oromethane/aceton mixture over glasswool in a second

testtube (see remark 3). Rinse the first testtube with resp. 2 and 1 ml dichloromethane/aceton mixture and combine, after filtering, the three portions.

Evaparate at 40°C the combined portions under a nitrogen stream and r

e-dissolve the residue in 50 ~1 methanol. Use again the Vortex mixer.

5.4 HPLC

5.4.1 Preparatien of standard graph

Prepare a standard curve by injecting 20 ~1 of aflatoxin

n

₁ salution containing resp.: 0.05; 0.1; 0.2; 0.4; 0.8 and 1.6 ng

n

_{1 •}

Platting the injected quantities against the area of the obtained

af-latoxin

n

_{1 respons} should result in a straight line.

5.4.2 Determination

Inject 20 ~1 of the sample extract (5.3) into the HPLC system. Campare retentien times for identification purpose. Calculate the aflatoxin n₁

content from the standard curve.

5.5 Remark

1. Drying the ether with metallic sodium is essential because in the

presence of water aflatoxin

n

₁ will elute in tl1e first direction.

2. After development of the thin layer plate in the first direction, befare cutting the plate, the spot of aflatoxin

n

₁ should be loc a-lized.

3. It is of great importance that no pieces of silicagel enters the

extract (if so adsorbtion resulting in a bad recovery will occur).

The use of a pipet tip (e.q. Finn tip no. 62) is recommended.

-(

(

Literature

1. N.D. Davis, U.L. Diener: J.AOAC ~ (1980) 107-109.

2. C.H. Thorpe, G.N. Hare, A.E. Pohland: V International IUPAC Sympo-sium on Nycotoxins and Phycotoxins, Vienna, september 1-3, 1982

page 52-55.

3. L.G.N.Th. Tuinstra, W. Haasnoot: Journal of Chromatography, 282 (1983) 457-462.

! .

DETERMINATION OF

AFL~TOXIN

_B1

1 • i I

I

Purpose and Scope

.

.

! ;

I

, I

Ann

e

x 2

The method makes it

p~ssible

to determine

.

the level of aflatoxin

B

₁

in

all animal

feed~ng st~ffs.

The lower limit of determination

is O.,g/kg (0.4ppb)

I

·

'

2.

Principle

The sample is extracted with a mixture of chloroform and water.

silica

The extract is then purified by using a disposable

~ep-Pak/cart

-ridge.

Aflatoxin B

1

is determined by HPLC.

Confirmatien by

conversion to aflatoxin B

₂₈

is described.

3.

Reà.gents

NB:

All the reagents must be of analytical reagent

1

_quality.

CAUTION: Chloroform is a possible carcinogen.

3.

1 Chloroform

3.2

Celite

545

filter aid

.

3.3

Aflatoxin B

₁

standerd

(5

~g

B

₁

/ml)

3.4

Toluene

3.5

Methanol

3.6 Chloroform - methanol mixture: Mix

97

m1

chloroform

wi~h

3

ml of methanol.

3.7 Mobile phase for HPLC- Water-saturated toluene-ethyl

-

acetate - formic acid

(85+25+5)

3.8

Toluene - acetonitrile

(98+2)

3.9

Trifluoroacetic acid

4.

Apparatus

4.1

Wrist-action shaker

4.2

Grinder-mixer

_. ~

4.4

4.5

4.6

4.7

4.8

Filter papers GF/A or

equivalent,15

cm

diam€ter

Rotary evaporator,

, wi th a 500 ml round-bottorn flask

Suitable liquid

dhr~matograph

.

I

HPLC columns

-

stainless steel 250 x 4.6 mm

id,

packed with

I

I .

5

~

Partisil 5

rr

:

5

~

Spherisorb ODS

Sep-Pak silica disposable cartridge

5.

Procedure

5.1

Preparatien of the sample

Grind the sample so that the whole of

it

will pass through a

sieve with a 1 mm mesh.

5.2 Extraction

Weigh 50

g

of finely ground feed

into

a 500 ml conical flask (4.3)

and add 250 ml of chloroform (3.1)

followed

by

25 ml

of distilled

water.

Stopper

t~e

flask and shake (4.1) for 30 minutes.

Filter

the extract through

'l 0

g

Cel i te 545 filter aid ( 3. 2) in a folded

glass fibre filter paper (4.4). Collect two 50 ml portions of

filtrate.

Add 10 )ll of aflatoxin standard (3.3) to one portion.

Evaporate both portions to

near

dryness using the rotary evaporator

(4.5)

5.3 Sep-Pak clean-up

Add extract to cartridge by using

two

0.5 ml portions of toluene

(3.4)_

.

Wash with 10 ml toluene. Elute aflatoxin B

₁

with 10

m1

of

chloroform-methanol (3.6) (fraction II). Evaporate fraction II to

near dryness and transfer to a small vial with chloroform (3.1).

~.4

High performance liguid chromatography

Set the fluorescence detector (4.6) to excitation 365 nm (15 nm

slit), emission 425

n~

(20 nm slit). Stabilise the system at a

flow rate of 2.5 ml/min. of mobile phase (3.7). Dilute

the

aflatoxin standerd solution

(3.3)

with toluene/acetonitrile (3.8)

to give

~~olution

_{containing 0.2;fg B1/ml._ Inject}

~~,fl

and

..

.

{

~---I

adjust the

sensitivit,~

of the fluorescence detector (4.6) to

give approx.

60

-

80% FSD.

Evaporate fraction

II

to dryness, dissolve

1

I

lt

:

.

"

il (

8)

d

i

t

0

1

c

in

0.5

ml

to uene ace

on~t~

e

3~

an

njec

5

~

.

ompare

peak heights obtained from the sample and the spiked extracts to

calculate the

concen~ration

of aflatoxin

B

1

in the sample.

5. 5 Confirmatien of the

_J~nti

ty

of

aflatoxin

B 1

a) Change the fluorescence excitation wavelength to

330

nm and

sequentially inject

50

fl of a solution containing

0.2

fg

_B1

/ml

followed by

50

)ll

of sample extract. Check that the peak height

ratio

365/330

nm

obtained with the sample extract agrees to within

10%

of that obtained with the standard solution.

b) Evaporate the extract

t~

dryness, add

50

~1

of trifluoro acetic

acid

(3.9)

and

exam~ne

a.gain by

HPLC.

Aflatoxin

B

₁

is thus

con-verted to aflatoxin

B2a

which has a longer

reten~ion

time than

aflatoxin B

1

under the

HPLC

conditions described.

6.

Observation

This method has been adaptcd from the multimycotoxin screening

procedure described by

M V

Howell and

P W

Taylor in

J.

Assoc. Off.

Anal.

Chem., 1981, 64 No 6

·

1:;56.

·

N

T CROSBY

LABORATDRY OF THE GOVERNMENT CHEMIST

AUGUST 1983

..

Vor

s

chJ

ag

der deutschen D

e

lega

t

jon

Juni

1984

Bestimmung von Aflatoxin

B

₁

mit GPC-Reinigung

1

.

Z\'Jeck

und Am·1endungsbere

i

eh

Ann

ex 3

Di

e

Methode

erlaubt

die

Bestimmun

g

von

Afla

t

oxin B

₁

in

Futtermitteln

1

die

Citrustrester enthalten. Si

e

ist auch für citrustresterfreie Futtermittel ge

e

i

g

n

et

.

?

Die

untere Grenze der

Bestimmbarkeit

beträ

g

t

~

_

m

G

/l

~

h

2

.

Prinzip

I

Die

Probe wird mi t Chloroform extrahiert.

Der Extral<t

\'lird

fil triert

und

ein aliquoter

Teil

am

Rotationsver-dampfer zur

Treekene

eingedampft.

Der

Rückst

a

nd \':ir

d

in

einer definierten

Menge

einer Cyclohexan/Essi

g

s

ä

ure-ethylester-Mischung

aufgenommen

1

hierven werden

5

ml

auf eine

GPC-Säule

ge

g

eben

und

mit einer Cyclohexan/

Essigsäureethyle~ter-Mischung

eluiert.

Die

aflatoxin-hal tige

Fral{tion vlird

aufgefangen

1

eingeengt

und

zvJ

e

i-dimensional auf einer DC-Platte aufgetrennt, d

as

Afl

a

tox

i

n

B

1

v.rird

fluorodensi

tornetris

eh

bestimmt.

3

.

Reagentien

3

.1

Chloroform,

p.a

.,

phosgen- und

säurefrei

3.2

Cyclohexan, p.a.

3

,

3

Essigsäureethylester

1

p.a.

₁

3.4

Aceton,

p.a.

3

.

5

Ameisensäure, p.a., 100

%ig

3.6

Toluol, p.a.

3.7

Celite

545

3

.

8

J

1ischung aus

Cyclohexan (3.2) und

Essigsäureethylester (3.3)

1

:

1

(V/V)

3. 9

Laufmi

ttel

1

: Jv1ischung

aus Chloroform

(

3. 1)

und Aceton

(3.4)

9

:

1 (V/V)

-3.10

Lauîmittel

2

:

MischW1g

aus Toluol

(3.6),

Essigsäureethylester

(3.3) und

J~eisensäure

(3.5)

5

: 4

:

1 (V/V/V/)

4.

Geräte

4.1

I-1ahlgerät

(~1ahlfeinheit

mindestens 0,5

rnm)

4.2 Schüttelmaschine

4.3

Faltenfilter (Macherey-Nagel

614

1/4

oder

gleiclw1ertige Quali tät)

4.4 Chromatographierohr

aus Glas für

Gelpermeation,

Länge der

Gelperm~ationsfüllung:

ca

.

35 cm,

irnerer Durchmesser ca. 2,5 cm

4.5

4.6

4.7

4.8

Füllung:

Biobeads S-X

III oder

gleichv1ertiges

Material

Vakuum-Rotationsverdampfer mit

500-ml-Rundkolben

500-ml-Erlenmeyerkolben oder RW1dkolben mit Schliff

J\usrüstW1g für Dünnschichtchromatographie

(Fertig-

jdie

(glaspiatten für Dünnschichtchromatographie

20 x 20 cm

,

Fa.

Baker

·

Si

250 oder gleichwertiges

Material

4.9 UV-Analysenlampe, Bereich

365

nm

4.10

BetrachtW1gsgehäuse zur

Aufnahme der

UV-Analysen-lampe

sowie

der DG-Platten

.

/

·

L·.11 F•luor.,odensJ.'·'-ometer mJ.'t

•

~

Schreiber bzw. Integrator

4.12 Membranfilter 0,45 um (regenerierte

Zellulose)

4.13 10-ml-MeBkolben

5. Ausführung

5

.1

Vorbereitung der Probe

Die

Probe

wird

gemahlen,

so

daB sie

vollständig

durch

ein

0,5-mm-Sieb hindurchgeht

..

5

.

2

Extraktion

50,0

g

der

gemahlenen

und homogenisierten

Probe

werden in einen 500-ml-Erlenmeyerkolben oder

Rundkolben (4.6) eingewogen, man

fügt 25 g Celite

545

(

3.7)

hinzu und

vermischt

mit einem Glasstab;

-I I I

/

I

I

I I

₃

~

0

J

J

/v

1

/I

v

-

.tJ

dann gibt

man 25 ml \·lasser hinzu, vermischt

nochmals mit dem Glasstab und fügt schlief3lich

250

ml Chloroform (

3.

1) hinzu. Der Kol ben \':ird

verseblossen und 60 Hin

.

mit

der

Schüttelmaschine

(4.2)

geschüttelt.

..

Anschlief3end \·Jird durch

ein

Paltenfilter

(4.3)

filtriert

.

Die ersten 10

ml

des

Filtrats werden verworfen, der Rest wird

auf-gefangen

.

5.3

Gelpermeationschromatographische Reinigung

Auf dem Jllarkt \'Jerden sm·1ohl

automatische

Geräte

als auch

Fertigsäulen

zur

gelpermeationschrornato-graphischen

Reinigung angeboten

.

V

on

dem nach 5

.

2 gev10nnenen

lli:trakt wird

ein

rnög-lichst grof3er altquoter

Anteil am

Rotationsver-darnpfer

(

4.

5)

zur Trocl<ene eingedampft.Der Trocl-;:enrückstand

wird mit der Mischung Cyclohexan/Essigsäureethylester (3

.

8)

in

Lösung

gebracht

und quant

i

tati v in

einen

1 O-rnl-1"1e

Bkolbel)

(Hl

übergeführt; mit der Mischung (3

.

8)

wird

zur

Marke

a

uf

ge

- ·

füllt

.

Man filtriert durch

ein

Membranfilter(4

.

12)w1d

g

ibt

von

diesem

Filtrat

5

ml

entweder mittels eines

Pro-bengebers bzw. mittels

einer Spritze auî die

GP

C

-Säule

(

4

.

4)

.

l

1li

t

dern Laufmi ttel Cyclohexan/Essi

g

-säureethylester

(

3

.

8)

\

·lird

bei

e in

Er (

Dw;cmluf3geschv.rindig-(ke~t

von

5

ml/~in.

eluiert

.

Die Fraktien der

ersten 32 Min

.

(ca

.

160 ml)

\

·lerden als

Vorlauf vervlOrfen

.

\'

!ähre

nd

der

nächsten 12 Min

.

(ca

.

60

ml) wird

eine

Fr

aktio

n,

die das gesamte

Aflatoxin

B

₁

enthält, gesamoelt

.

20

I'iin

.

(

entsprechend

100 ml) werden für

das

H

ac

h-waschen

angesetzt.

Diese

aufgeführten Zahlen

si

nd

als

Richtdaten

zu

\'Jerten.

Sie

müssen

für jede Säule

mi

ttels

eines

Eichstandards

überprüft

.

\

·Jerden

.

Die

durch Gelpermeationschromatographie

gewonnene

af

latoxinh

a

l

tige

Fraktien v1ird

am

Rotationsverdamp

fer

(

4

.

5) zur Treekene eingedampft

und

anschlief3end

mi t

4

5

.

4

Z\·

J

eidimensionale Dünnsc

h

ichtchromatographie

Hier wird

gena\Jso

verfahren

\·lie

in der Juntlichen

t

·

1ethode 13 unter Punkt

5

.

4

angegeben (

s.

a.

Methoden-b

u

ch

III

,

Kap

.

1lJ

_..

.1

.2),

es sind jedoch die

Lauf-mittel

Chloroform/Aceton

(

3

.

9)

in der ersten Laufrichtung

W1d

Toluol/Essigsäureethylester/Ameisensäure

(3.10)

in der

zwei

ten Laufrichtw1g

zu

benutzen

.

Je nach Aflatoxin-Gehalt köru1en bis z

u

200

~1

des

nach

5

.

3

gevJOnnenen

Extraktes

auf

die DC-Platte

suf-getragen

werden

,

Die Platte muf3

sowohl

nach der ersten Entwicklung

als auch na

eh

der zHeite

n

Ent

\

'!ic

l

<lung

(

vor der

Fluoreszenzmessung) völlig lösungsmittelfrei

sein

(

ggf

.

Vakuumtrocl<enschrank bei Zimmertemperatur

be-nutzen)

.

Quantitative

AuswertunJd~fbhDC-Platten

Die

Aus

\

·Jertung

erfolgt fluorodensi

tometrische J.1essw1g,

\'IObei

Stándardflecke

mi

t

be

l

<annten Aflatoxin

B

1

-

1

•1engen

auf

derselben Platte

als

J.1ef3gröf3e zugrunde zu legen

sind

.

Unabhängig hierven empfiehlt

es sich

,

auf

einer

anderen Platte der gleichen Charge

eine

ent-sprechende

tiJenge an

Aflato::~in B

1

als

Standard Z\·.'e

i-dimensional dünnschichtchromatog;raphisch

zu

unter-suchen

und die

Fluoreszenzintensi tät

des

Z\

·

J

eidimensional

entv.•ickel

ten

Aflatoxin B

₁

-Fleckes

zu

messen

.

Erfah-rungsgemäf3

sind die

Unterschiede bei den unter 4.8

genarmten

DC-Platten

gleieb9P CkaliPgf2

so gering,

daB

die

Fluoreszenzintensität nach zweidimensionaler

Chromatographie mit derje

n

igen

nach

eindimensionaler

Chromatographie übereinstimmt

(

Lösungsmittelfreiheit

der Platten vorausgesetzt)

.

.

. ·

Annex 4

Amendment to the EEC method of determination of aflatoxin Bl (

76/372/E~F)

L lo2 1976

.

Determination of Aflatoxin B

₁

by HPLC.

B.T.Viuf, Statens Foderstofkontrol

1.

Purpose and scope.

The method makes it possible to determine aflatoxin B

₁

in

feedingstuffs. The lower limit of determination is

1

pg/kg.

2. Principle

.

Instead of thin layer chromatography add

:

The ammount of aflatoxin

is determined by HPLC-chromatography and measurement of fluorecence

in a packed flow-cell

.

3 Reagents.

add

.

3.19

HPLC- solvent. Prepare from water-saturated methylenechloride,

cyclohexane , acetonitrile (loo+3o+4) a mixture containing about

o,5% absolute ethanol. The amount of etha

n

ol can be varied to obtain

optimum resolution of aflatoxin B

₁

• Allreagents must be HPLC-grade

.

3.2o

Prepare in chloroform standardsolution containing o.2, o.l, and

o.o5 pg/ml aflatoxin B

₁

according to point 7.

4. App

e

ratus

4.13 High pressure liquid chromatograph

.

Apperatus equipped with

injektor, normal phase column (Spherisorb S 5

W

5

p

m or equivalent

,

loo x 4.6 mm ), fluorecence detector equipped with a flowcell

packed with

l

ichrosorb 6o silica gel (3o

pm)

₁

emmision 365 nm and

excitation 425 nm. Use a flow rate l.o ml/min

.

5.

Procedure

Add instead of 5.4 and 5.5:

New 5.4

Separation and detection.

Prepare a standard curve by injecting 2o pl of aflatoxin B

1

standards containing resp.: o.2

,

o.l

,

and o.o5 pg/mi:- Inject

2o pl of the sample extract dissolTed in chloroform (5.3). Cal

' '

.

..

.. 8.22 8.36 11.61 13.+4 ST~ ST~ • .1 • _6s.t ( ~R/!5/SJ . ... :5J::D. A.c(A~ RU4. ~6 ~PR/15/33

RT

_{AREA TYPE} 15:<41:<4~

8.22 _~lat

_PB

...

AR/lfl'

AREA).,

ARE~

1.58. tl9e

RT

AREA TY~ M/HT

8.36

_{7291998 88 9.-418 92}- ARE~ _.878

·--

11

.

61

'4-R9389 88 8.567

~~"~

~

57.122

-=~~~~~~==-~

. . tb,.,

'

a

.

6alb,

11.93 ~/ 12.87

q

~ STOP

RUH I

37 STOP AREA%

RT AREA TYPE AfUHT AREA",

9.44 499930 py _{9.288 0.} 30~

I

1. 89 3913790 yp _9.814 2.91/ 2.91 3334999 py _9.927 3.22~ j

RUH I

97 SEP/16/82 11:48:99 4.76 4392699

w

l. 359 4.251

I

8.69 2.9615[+97 yy _9.691 28.66~ AREA% 9.88 1.6144E+07 yy 9.673 15. 62~

I

RT AREA TYPE AfUHT AREA% 11.93 2.9597E+97 yy 9.644 27. 67~

I

5.29 324829 py _9.329 12.87 1. 7824[+97 yy 9.921 17.251 I 1. 491

I

6.64 3779999 VY 9.885 17.191 I 8.5<4 1 . 9<475[ +97 YB 9.661 47.756 J 19.92 5822399

BB

9.?26 26.543 • r -• ,.. ... ,... ... -, ,."-,n

Annex 5

In the first month's of 1984 five samples of cattie feed intended for dairy were five times at random on several days analysed with the HPLC

method in order to estimate the reproducibility. The analyses were carried out by a technician on blind samples. In table I the results are given.

Table 1

Hean content of v.c.(R) Range

five analysis (HPLC)

).lg/kg % ).lg/kg 2,2 93 0,5- 3,4 4,5 32 2,5- 5,4 8,0 12 6,8- 9,1 10,2 18 8,8-13,5 11,1 10 9,6-12,3

At a level of 10 ).lg/kg a realistic coäfficient of variation of less than 20% can be achieved.

Draft .13

' I •• 1 f Annex 6

1

th collaborative study

lab

N2

_{aflatoxin B}

₁

1

2

,

5

2

2,0

3

2,4

4

9,0

x)

5

2,5

_x)

=

outlier

6

2,9

7

3,0

8

24,0

x)

9

2

,

0

10

2,0

11

1,

7

N

₉

x

=

2

,

3

ppb

s

0,44 ppb

V

(S%)

=

1

8,9

%

2nd

collaborative stu

d

y

lab

N2

af

l

_{atoxin B}

₁

( I

_ppb

I

1

6,9

2

7,7

3

7,5

4

6,6

5

6,3

N =

₅

x

=

_7,0

ppb

s

0,59 ppb

V (S%) =

8,5

%